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ABSTRACT: Platelet factor 4 (PF4) is only expressed in platelets and is an appropriate marker for studying megakaryocytic differentiation. We previously characterized cDNA and genomic clones for human PF4 (hPF4) and now present transient expression studies defining the promoter of the gene. 12-O-tetradecanoyl-phorbol-13- acetate (TPA) induces megakaryocytic differentiation of human erythroleukemia (HEL) cells, providing an excellent model system for the study of megakaryocyte-specific promoter activity. Luciferase reporter-gene constructs containing sequences from -2074 to +49 were used to map regions that may regulate PF4 gene expression. The sequence in the region -239 to -107 increased basal promoter activity by four- to five-fold in TPA-induced HEL cells. The sequence between -239 and -107 contains 53 consecutive thymidine residues. Functional studies using constructs in this region show that poly(T) and the region -187 to -107 are necessary for the total increase in activity in TPA-induced HEL cells. Mobility-shift assays show that the poly(T) tract binds TPA-inducible proteins. The results suggest a complex promoter for the PF4 gene involving a basal nonspecific promoter element between -107 and +49, a positive promoter element between -239 and -107 binding specific nuclear proteins from megakaryocyte-lineage cells, and a silencer-like region between -2074 and -1653.
Experimental Hematology 02/1995; 23(1):49-57. · 2.90 Impact Factor
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Nucleic Acids Research 11/1990; 18(19):5919. · 8.03 Impact Factor
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ABSTRACT: Platelet factor 4 (PF4) is a 70 amino acid heparin-binding protein released from the alpha-granules of activated platelets. Its exact biologic function is not known, although PF4 is a member of a multigene family involved in chemotaxis, coagulation, inflammation, and cell growth. We previously cloned the cDNA for human PF4 from a human erythroleukemic (HEL) cell expression library. We now report the isolation and sequence determination of the gene for human PF4. This gene contains three exons and spans approximately 1,000 basepairs (bp). Concurrently, we have cloned a highly homologous gene that we have called PF4alt. We show that PF4 and PF4alt are non-allelic genes: the human PF4 gene is encoded on a 10 kilobasepairs (kb) EcoRI fragment, and its DNA sequence agrees with protein and cDNA data for PF4, while PF4alt is encoded in a polymorphic 3 or 5 kb EcoRI fragment. Compared with PF4, this gene has 14% DNA and 38% amino acid divergence in the signal peptide region, and 2.6% DNA and 4.3% amino acid divergence in the coding region of the mature protein. PF4alt contains three amino acid substitutions (P58----L, K66----E, and L67----H) near the C-terminus, in a region known to be critical for PF4 function. Primer extension studies show the 5'-untranslated region of PF4 is 73 bp long. A TATA box is present 30 bp 5' to the transcription start site. A 90 bp stretch of pyrimidines (including 53 consecutive thymidine residues) begins at -227 bp and is analogous to a similar region of 30 residues 5' to the rodent PF4 gene. This pyrimidine-rich region is absent from the PF4alt gene; however, DNA homology exists between the two human genes in the 5'- and 3'-flanking regions and extends for over 3.6 kb. Alternating purine/pyrimidine tracts occur both 5' and 3' to PF4 and PF4alt but do not define the endpoints of the gene duplication, which extend beyond these sequences at least at the 5' end. Northern blot analysis using gene-specific oligonucleotides and platelet RNA showed an 800 or 900 nucleotide (n) message for PF4 and PF4alt, respectively. Northern blot and primer extension studies show that steady-state platelet PF4 mRNA levels are approximately one magnitude greater than PF4alt mRNA levels. Thus, these studies demonstrate that PF4alt mRNA is expressed in platelets. Whether PF4alt protein is expressed remains to be determined, and the nature of its biologic function needs to be studied.(ABSTRACT TRUNCATED AT 400 WORDS)
Blood 08/1990; 76(2):336-44. · 9.90 Impact Factor
